Method for Using Electrochemical Bioreactor Module with Recovery of Cofactor

ABSTRACT

Provided herein are composition and process for using an electrochemical device for the reduction of the oxidized state of phosphorylated or non-phosphorylated nicotinamide adenine dinucleotide to the reduced state in which unwanted products of the electrochemical reduction are recovered as the oxidized state of the phosphorylated or non-phosphorylated nicotinamide adenine dinucleotide and returned to the electrochemical device for reduction.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application of U.S. applicationSer. No. 16/084,579, filed Sep. 13, 2018, now U.S. Pat. No. 10,927,395,which is a U.S. National Phase application under 35 U.S.C. 371 ofInternational Application No. PCT/US2017/022241, filed Mar. 14, 2017,which claims priority to and the benefit of U.S. Provisional ApplicationNo. 62/308,175 filed Mar. 14, 2016, the disclosure of each of which isincorporated herein by reference in their entireties.

TECHNICAL FIELD

The present disclosure relates generally to the use of biologicallymediated reactions that alter the oxidation state of compounds, andspecifically the oxidation state of carbon atoms in a given chemicalcompound.

More specifically, the present disclosure relates to an improved methodof utilizing the Electrochemical Bioreactor Module (EBM) in whichundesired forms of nicotinamide adenine dinucleotide cofactor which areproduced during the electrochemical reduction of the oxidized form ofthe nicotinamide adenine dinucleotide cofactor are recovered forrecycling through the EBM.

BACKGROUND

The use of enzymes which perform a reduction and are capable of reducinga provided substrate to a desired reduced product, has been widelyreported. (Dodds et al, J. Am. Chem. Soc., (1988) 110(2), 577-583; Doddset al, Proceedings Chiral Europe'95. London, (1995), 55-62; A. Liese etal, Appl Microbiol Biotechnol (2007) 76:237-248; Liese, A.; 2nd ed.Enzyme Catalysis in Organic Synthesis, (2002), 3, 1419-1459; Kula, M.R.; Kragl, U. Stereoselect. Biocatal. (2000), 839-866; Chartrain, M.;Greasham, R.; Moore, J.; Reider, P.; Robinson, D.; Buckland, B. J. Mol.Catal. B: Enzym. (2001), 11, 503-512; Patel, R. N. Enzyme Microb.Technol. (2002), 31, 804-826; Patel, R. N. Adv. Appl. Microbiol. (2000),47, 33-78; Patel, R. N.; Hanson, R. L.; Banerjee, A.; Szarka, J. Am. OilChem. Soc. (1997), 74, 1345-1360; Hummel, W. Adv. Biochem. Eng.Biotechnol. (1997), 58, 145-184; Whitesides et al, Appl. Biochem. andBiotech. (1987) 14, 147-197; Whitesides et al, Biotechnology and GeneticEngineering Reviews, Vol. 6. September 1988).

To accomplish such a reduction of a provided substrate, electrons mustbe provided to the reaction. In biological systems, both in vivo and invitro, these electrons, generally called “reducing equivalents”, areprovided by small molecules generally termed “cofactors”. The mostcommon cofactor is nicotinamide adenine dinucleotide, NAD. Aphosphorylated form of the cofactoralso exists, and both forms providereducing equivalents to the enzymes that catalyze reactions requiringreducing equivalents.

The nicotinamide adenine dinucleotide cofactors exist in an oxidizedstate and in a reduced state, and these are respectively abbreviated asNAD(P)+ and NAD(P)H in the open literature. However, these abbreviationswill be avoided here.

Most generally, the enzymes accepting reducing equivalents from thereduced state of the nicotinamide adenine dinucleotide cofactors aretermed “oxidoreductases” and are classified by the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology as EC 1.n.n.n. Of particular interest are the enzymes generallytermed “dehydrogenases” or “ketoreductases” in the classes EC 1.1.n.nand EC 1.2.n.n, as well as those termed “mono-oxygenases” in the classesEC1.13.n.n and EC.1.14.n.n. This last group of enzymes are also termed“P450” enzymes or “CYP” enzymes.

When performing reactions catalyzed by such enzymes usingmicro-organisms growing on a carbon source such as a carbohydrate, thereducing equivalents are generated by oxidizing a portion of thecarbohydrate to CO₂, that is, some of the carbohydrate provided to themicro-organism is sacrificially oxidized in order to provide electronsfor the micro-organism to use in various metabolic processes. While theresulting electrons are desirable and useful to the microorganism, thecarbon atoms sacrificed by oxidation to CO₂ are lost. It is alsopossible to provide carbon sources other than carbohydrates that arealso sacrificed to produce the desired reducing equivalents. It will beclear that while such an in vivo process may perform the desiredreaction catalyzed by the oxidoreductase enzyme, recovering the desiredproduct from the milieu of the in vivo system, e.g. a fermentationbroth, can be difficult. It is thus advantageous to utilize an in vitrosystem for performing the desired reaction.

It is possible to isolate the needed oxidoreductase enzyme and use it tocatalyze reactions in vitro. Significantly improved chemical processescould be achieved by an in vitro system which allows the use of theplethora of oxidoreductase enzymes in processes resembling standardcatalytic chemical processes. Such systems avoid the issues ofrecovering the desired product of the reaction from fermentation brothsand can provide further advantages by allowing the enzyme to be usedunder non-physiological conditions, and the use of a variety of methodsto immobilize or otherwise contain the enzyme, and allow it to be usedfor extended periods of time. Such immobilization or other containmentmethods also allow simple and efficient recovery of the desired productof the reaction from the enzyme system. A further advantage of usingisolated enzymes in a cell-free system is that multiple enzymes can beused, allowing a series of reactions to be performed.

Micro-organisms containing useful oxidoreductases are widely known innature and can be found quickly by simple screening (A. Zaks et al,Tetrahedron (2004) 60,789-797). If a particular oxidoreductase enzyme isrequired, the enzyme can be readily cloned and over-expressed in astandard industrially useful host such as S. cerevisiae or E. coli, andisolated by standard methods, and used in an in vitro system.

However, the reducing equivalents must still be provided to such invitro systems. Analogous to in vivo systems, a sacrificial substrate canbe provided, which is oxidized. This generates the necessary reducedstate of the nicotinamide adenine dinucleotide cofactors for providingreducing equivalents for the desired reaction.

It will be clear that the need of a sacrificial substrate removes atleast some of the advantages of an in vivo system, since a secondproduct, the product of the oxidation of the sacrificial substrate, mustnow be separated from the desired product. While formate may be usedsacrificial substrate, generating CO₂ as the result, this requires theuse of an additional enzyme, formate dehydrogenase, which may not beconvenient.

Thus it is most desirable to provide the electrons required for thereaction, that is, the reducing equivalents required by theoxidoreductase enzyme, in a manner that does not require sacrificialsubstrates or additional enzymes.

If electrons could be provided from an external source, that is, anelectrical current, then the need to provide a sacrificial substrate toprovide electrons would be eliminated, and oxidoreductase enzymes couldbe used as conventional catalysts, performing reactions without the needfor living cells or associated enzyme systems or processes.

This situation has been recognized by others, and a number of attemptsto deliver electrons to biological systems by electrochemical methodshave been published.

It has been reported by Park and Zeikus in J. Bacteriol. 181:2403-2410,1999 that the compound called Neutral Red would undergo reversiblechemical oxidoreductions with the nicotinamide adenine dinucleotidecofactor, that is, Neutral Red in its reduced form (NR_(red)) has asufficiently low redox value that it will transfer electrons to, andthus reduce, the nicotinamide adenine dinucleotide cofactor from itsoxidized state to its reduced state. In this process, the Neutral Redbecomes oxidized to the species NR_(ox) which is then available toaccept an electron from the cathode and thus return to the reduced formNR_(red), which is in turn available to again reduce the oxidized stateof the nicotinamide adenine dinucleotide cofactor.

U.S. Pat. No. 7,250,288 B2 to Zeikus et al. discusses the need forimproving electrode efficiencies in electrochemical bioreactor systemand proposes improvements such as linking the nicotinamide adeninedinucleotide cofactor, Neutral Red, and the enzyme fumarate reductase tothe electrode in order to improve electron transfer characteristics.While the above may improve electron transfer characteristics, it mayalso be advantageous to improve upon the electrochemical bioreactorsystem design and its use in other ways such as those described below.

The requirements for providing reducing equivalents by electrochemicalmethods are disclosed in PCT publication No. WO2014039767 A1,“Electrochemical Bioreactor Module and Methods of Using the Same”. Theuse of isolated enzymes in conjunction with the electrochemicalreduction of the oxidized form of nicotinamide adenine dinucleotidecofactor to its reduced form, and considerations to be made, aredisclosed in PCT publication No. WO2016070168 A1, “ImprovedElectrochemical Bioreactor Module and Use Thereof”.

When the oxidized state of the nicotinamide adenine dinucleotidecofactor is electrochemically reduced, several different reduced speciescan result. These species differ only in the position on thenicotinamide ring at which the reduction has formally occurred. This canbe at the 2-position, the 4-position or the 6-position on thenicotinamide ring, and these species are termed the 1,2-dihydro-NAD,1,4-dihydro-NAD and 1,6-dihydro-NAD isomers respectively. (Chakravertyet al, Biochem. Biophys. Res Comm., (1964), 15(3), 262-268; Chakrevertyet al, Jour. Biol. Chem (1969), 244(15), 4208-4217). These are shownbelow in Illustration A. The carbon atoms of the nicotinamide ring arenumbered in the oxidized form, with the nitrogen atom as position 1. Thedesired reduced product is the 1,4-dihydro-NAD isomer, commonly called(3-NADH, and this is the biologically active form of the co-factorrequired for most dehydrogenase, ketoreductase and P450 enzymes. Theother isomers, 1,2-dihydro-NAD and 1,6-dihydro-NAD do not have knownuseful biological activity with dehydrogenase, ketoreductase or P450enzymes.

A fourth species, a dimer of the NAD cofactor, can also form during theelectrochemical reduction of the oxidized state of nicotinamide adeninedinucleotide. This is termed the 4,4′-dimer and is also shown inIllustration A. Like the 1,2-dihydro-NAD and 1,6-dihydro-NAD forms ofthe cofactor, the dimer has no known useful biological activity withrespect to oxidoreductase enzymes, and cannot deliver reducingequivalents to them (Burnett et al, Biochem. (1968), 10(7), 3328-3333;Biellmann et al, Tetrahedron Letters (1978) 7, 683-686; Kirkor et al,Eur. J. Biochem. (2000), 267, 5014-5022).

Illustration A

In a system where an initial amount of the oxidized state of thenicotinamide adenine dinucleotide cofactor is electrochemically reducedfor the purpose of providing reducing equivalents to an oxidoreductaseenzyme, the desired, biologically active 1,4-dihydro-NAD isomer will beformed and will provide reducing equivalents to the oxidoreductaseenzyme (or enzymes) in the system, and be oxidized back to the oxidizednicotinamide adenine dinucleotide cofactor, ready for another cycle ofelectrochemical reduction. However, the two non-biologically activeisomers, the 1,2-dihydro-NAD and the 1,6-dihydro-NAD, as well as the4,4′-dimer initially produced by the electrochemical reduction of theoxidized nicotinamide adenine dinucleotide cofactor will not be consumedand will simply remain in the system. The oxidized nicotinamide adeninedinucleotide cofactor resulting from the productive delivery of reducingequivalents to the oxidoreductase enzyme will be again reduced to give amixture of the three isomers, plus the dimer, as in the previousreduction process. In this cyclic manner, the amount of oxidizednicotinamide adenine dinucleotide cofactor available for electrochemicalreduction decreases continually, and after a short period of time,essentially none is left. This problem was recognized four decades ago(Whitesides et al, Appl. Biochem. and Biotech. (1987) 14, 147-197;Whitesides et al, Biotechnology and Genetic Engineering Reviews, Vol. 6.September 1988).

Thus, it is useful to remove the undesired 1,2-dihydro-NAD and the1,6-dihydro-NAD isomers as well as the 4,4′-dimer should they beproduced during the electrochemical reduction of oxidized nicotinamideadenine dinucleotide cofactor to the desired 1,4-dihydro-NAD species,(i.e. β-NADH). Further, it is useful to recover the 1,2-dihydro-NAD andthe 1,6-dihydro-NAD isomers as either as the desired 1,4-dihydro-NADisomer, or as the initial oxidized state of the nicotinamide adeninedinucleotide cofactor.

Provided herein are methods and systems for achieving the aboveobjectives, Other objectives, features, and advantages of the presentdisclosure will be apparent on review of the specification and claims.

SUMMARY

The present disclosure, in one aspect, provides a system forelectrochemically generating NAD(P)H₂ reducing equivalents, the systemcomprising:

-   -   (a) an electrochemical cell comprising an anode contained in an        anode chamber and a cathode contained in a cathode chamber;    -   (b) a first process stream containing NAD(P), passing through        the cathode chamber and continuously in contact with the cathode        from which electrons are transferred to the NAD(P) to produce a        second process stream containing reduced species 1,4-NAD(P)H₂,        1,2-NAD(P)H₂, 1,6-NAD(P)H₂, and [NAD(P)]₂, while optionally        producing hydrogen;    -   (c) a substrate of an oxidoreductase or P450 enzyme, which, when        contacted with the second process stream in the presence of the        oxidoreductase or P450 enzyme, is transformed to a product while        concomitantly consuming the 1,4-NAD(P)H₂ in the second process        stream and producing a first recovered NAD(P) and a third        process stream; and    -   (d) at least one of a renalase enzyme, a Mung Bean Phenol        Oxidase enzyme and illumination at a wavelength of about 254 nm        or exceeding about 320 nm, which, when contacted with the third        process stream, convert at least one of the 1,2-NAD(P)H₂,        1,6-NAD(P)H₂, and [NAD(P)]₂ therein to a second and optionally a        third recovered NAD(P).

In some embodiments, the system includes the renalase enzyme forconverting the 1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ into the second recoveredNAD(P).

In some embodiments, the system includes the Mung Bean Phenol Oxidaseenzyme for converting the 1,2-NAD(P)H₂, 1,6-NAD(P)H₂, and/or [NAD(P)]₂into the second recovered NAD(P) and/or the third recovered NAD(P).

In some embodiments, the system includes the illumination for convertingthe [NAD(P)]₂ into NAD(P).

In some embodiments, the system includes the renalase enzyme and theMung Bean Phenol Oxidase enzyme. For example, the third process streamcan be contacted with the renalase enzyme resulting in conversion of the1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ into the second recovered NAD(P) and afourth process stream, wherein the fourth process stream can becontacted with the Mung Bean Phenol Oxidase enzyme to convert the[NAD(P)]₂ therein to the third recovered NAD(P).

In some embodiments, the system includes the renalase enzyme and theillumination. For example, the third process stream can be contactedwith the renalase enzyme resulting in conversion of the 1,2-NAD(P)H₂ and1,6-NAD(P)H₂ into the second recovered NAD(P) and a fourth processstream, wherein the fourth process stream can be contacted with theillumination to convert the [NAD(P)]₂ therein to the third recoveredNAD(P).

In some embodiments, the system includes the Mung Bean Phenol Oxidaseenzyme and the illumination. For example, the third process stream canbe contacted with the Mung Bean Phenol Oxidase enzyme resulting inconversion of the 1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ into the secondrecovered NAD(P) and a fourth process stream, wherein the fourth processstream can be contacted with the illumination to convert the [NAD(P)]₂therein to the third recovered NAD(P).

In some embodiments, the system can include, in one or more of theprocess streams, an electron transfer mediator (ETM) capable oftransferring electrons to NAD(P). The system can also include catalasefor decomposing hydrogen peroxide produced by the renalase enzyme, theMung Bean Phenol Oxidase enzyme, and/or the illumination.

Another aspect relates to a method for electrochemically generatingNAD(P)H₂ reducing equivalents, the method comprising:

-   -   (a) providing an electrochemical cell comprising an anode        contained in an anode chamber and a cathode contained in a        cathode chamber;    -   (b) passing through the cathode chamber a first process stream        which contains NAD(P) and is continuously in contact with the        cathode from which electrons are transferred to the NAD(P) to        produce a second process stream containing reduced species        1,4-NAD(P)H₂, 1,2-NAD(P)H₂, 1,6-NAD(P)H₂, and [NAD(P)]₂, while        optionally producing hydrogen;    -   (c) contacting the second process stream with a substrate of an        oxidoreductase or P450 enzyme such that the substrate, in the        presence of the oxidoreductase or P450 enzyme, is transformed to        a product while concomitantly consuming the 1,4-NAD(P)H₂ in the        second process stream and producing a first recovered NAD(P) and        a third process stream; and    -   (d) contacting the third process stream with at least one of a        renalase enzyme, a Mung Bean Phenol Oxidase enzyme and        illumination at a wavelength of about 254 nm or exceeding about        320 nm, thereby converting at least one of the 1,2-NAD(P)H₂,        1,6-NAD(P)H₂, and [NAD(P)]₂ therein to a second recovered NAD(P)        and optionally a third recovered NAD(P).

The method in some embodiments can further include contacting the thirdprocess stream with the renalase enzyme for converting the 1,2-NAD(P)H₂and 1,6-NAD(P)H₂ into the second recovered NAD(P).

In some embodiments, the method can further include contacting the thirdprocess stream with the Mung Bean Phenol Oxidase enzyme for convertingthe 1,2-NAD(P)H₂, 1,6-NAD(P)H₂, and/or [NAD(P)]₂ into the secondrecovered NAD(P) and/or the third recovered NAD(P).

In some embodiments, the method can further include contacting the thirdprocess stream with the illumination for converting the [NAD(P)]₂ intoNAD(P).

In some embodiments, the method can further include contacting the thirdprocess stream with the renalase enzyme resulting in conversion of the1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ therein into the second recovered NAD(P)and a fourth process stream, and further comprising contacting thefourth process stream with the Mung Bean Phenol Oxidase enzyme toconvert the [NAD(P)]₂ therein to the third recovered NAD(P).

In some embodiments, the method can further include contacting the thirdprocess stream with the renalase enzyme resulting in conversion of the1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ therein into the second recovered NAD(P)and a fourth process stream, and further comprising contacting thefourth process stream with the illumination to convert the [NAD(P)]₂therein to the third recovered NAD(P).

In some embodiments, the method can further include contacting the thirdprocess stream with the Mung Bean Phenol Oxidase enzyme resulting inconversion of the 1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ therein into the secondrecovered NAD(P) and a fourth process stream, and further comprisingcontacting the fourth process stream with the illumination to convertthe [NAD(P)]₂ therein to the third recovered NAD(P).

In some embodiments, the method can further include providing, in one ormore of the process streams, an electron transfer mediator (ETM) capableof transferring electrons to NAD(P).

In some embodiments, the method can further include providing catalasefor decomposing hydrogen peroxide produced by the renalase enzyme, theMung Bean Phenol Oxidase enzyme, and/or the illumination.

In some embodiments, the method can further include returning the thirdprocess stream to the cathode chamber.

In any of the systems and methods disclosed herein, the renalase enzymeand/or Mung Bean Phenol Oxidase enzyme may be recombinantly expressedfrom microorganisms such as Escherichia coli, Bacillus subtilis,Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris,and Bacillus megaterium.

The renalase enzyme in certain embodiments can be a mutant form(naturally occurring or genetically engineered) having a lower oxidationactivity for 1,4-NAD(P)H₂ than the wild type renalase enzyme, and/or ahigher oxidation activity for 1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ than thewild type renalase enzyme. In some examples, the mutant form can berecombinantly expressed from microorganisms such as Escherichia coli,Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae,Pichia pastoris, and Bacillus megaterium.

The Mung Bean Phenol Oxidase enzyme can be a mutant form (naturallyoccurring or genetically engineered) having a lower oxidation activityfor 1,4-NAD(P)H₂ than the wild type Mung Bean Phenol Oxidase enzyme,and/or a higher oxidation activity for 1,2-NAD(P)H₂, 1,6-NAD(P)H₂ and/or[NAD(P)]₂ than the wild type Mung Bean Phenol Oxidase enzyme. In someembodiments, the mutant form is recombinantly expressed frommicroorganisms such as Escherichia coli, Bacillus subtilis,Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris,and Bacillus megaterium.

Also provided herein is a process for providing electrochemicallygenerated reducing power via the reduction of an oxidized form ofphosphorylated or non-phosphorylated forms of nicotinamide adeninedinucleotide cofactor to a redox enzyme system for the purpose ofcatalyzing a transformation of a substrate molecule.

The process can include steps for preventing the loss of cofactormaterial to reduced forms that are not used by the redox enzyme systemfor the purpose of catalyzing a transformation of a substrate molecule.

The process can include the electrochemical reduction of the oxidizedstate of nicotinamide adenine dinucleotide cofactor to give a mixture ofthe desired 1,4-dihydro-NAD species with the undesired 1,2-dihydro-NADand 1,6-dihydro-NAD species, and the undesired 4,4′-dimer. The desired1,4-dihydro-NAD species is converted back to the oxidized form of thenicotinamide adenine dinucleotide cofactor in the course of providingreducing equivalents to the oxidoreductase enzyme which is present tocatalyze the transformation of the substrate molecule. The remainingmixture of the 1,2-dihydro-NAD and 1,6-dihydro-NAD species, the4,4′-dimer, together with the oxidized state of the nicotinamide adeninedinucleotide cofactor is acted upon by the renalase enzyme. This enzymeoxidizes the 1,2-dihydro-NAD and 1,6-dihydro-NAD species back to theoxidized state of the nicotinamide adenine dinucleotide cofactor in thepresence of oxygen, producing hydrogen peroxide in the process. The MungBean enzyme oxidizes the 4,4′-dimer back to two molecules of theoxidized state of the nicotinamide adenine dinucleotide cofactor, alsoby using oxygen and generating hydrogen peroxide. Thus all speciesoriginally produced by the electrochemical reduction of the oxidizedform of the nicotinamide adenine dinucleotide cofactor, both desired andundesired, are returned to the original oxidized state of thenicotinamide adenine dinucleotide cofactor and no cofactor material islost. The 4,4′-dimer is photo-active, and can be returned to theoxidized state of the nicotinamide adenine dinucleotide cofactor speciesby irradiation with the appropriate wavelength; this may be performed inplace of, or in conjunction with, the Mung Bean oxidase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates, in a schematic, the utilization of renalase, MungBean Phenol Oxidase, and optionally illumination to recover and recyclecofactor material from the mixture of the desired 1,4-dihydro-NADspecies plus the undesired 1,2-dihydro-NAD and 1,6-dihydro-NAD speciesand the undesired 4,4′-dimer which are formed via the electrochemicalreduction of the oxidized state of nicotinamide adenine dinucleotidecofactor. In FIG. 1, the 1,2-dihydro-NAD, 1,4-dihydro-NAD and the1,6-dihydro-NAD species of the nictotinamide adenine dinucleotidecofactor are named 1,2-NAD(P)H₂, 1,4-NAD(P)H₂ and 1,6-NAD(P)H₂respectively, while the 4,4′-dimer is named [NAD(P)]₂ and the oxidizedform is named NAD(P).

DETAILED DESCRIPTION

The current literature most commonly uses the descriptor “NAD(P)+” toindicate the oxidized state of the phosphorylated and non-phosphorylatedforms of the nicotinamide adenine dinucleotide, and “NAD(P)H” toindicate the reduced state. The reduced state of the phosphorylated andnon-phosphorylated forms of the nicotinamide adenine dinucleotide areproduced by adding two electrons and two protons to the oxidized state.This common nomenclature is misleading, as plain reading of the commondescriptors “NAD(P)+” and “NAD(P)H” show the oxidized state of thephosphorylated and non-phosphorylated forms of the nicotinamide adeninedinucleotide to be a positively charged species “NAD(P)+”, while thereduced state of the phosphorylated and non-phosphorylated forms of thenicotinamide adenine dinucleotide, “NAD(P)H”, is shown to have only onehydrogen added relative to the oxidized state. Neither of theserepresentations is true. The oxidized state of the phosphorylated andnon-phosphorylated forms of the nicotinamide adenine dinucleotide is aneutral molecule in which the nitrogen of the nicotinamide ring bears aformal positive charge with is balanced by a negative charge one of thedeprotonated phosphate linkages, thus forming an internal salt orzwitterion. The reduced state of the phosphorylated andnon-phosphorylated forms of the nicotinamide adenine dinucleotide hasformally accepted a hydrogen molecule, H₂, relative to the oxidizedstate, and this achieved by two one-electron transfers balanced by twoprotons. This is shown formally by the following balanced chemicalreaction:

NAD(P)+2H⁺+2e ⁻→NAD(P)H₂

The published molecular weights of the oxidized and reduced states ofthe non-phosphorylated form of the nicotinamide adenine dinucleotideindicate the reality of this reaction clearly, the molecular weightsbeing 663.43 Da and 665.44 Da respectively and the difference being themolecular weight of a single hydrogen molecule, H2.

In the present disclosure, the descriptor “NAD(P)” indicates theoxidized state of the phosphorylated and non-phosphorylated forms of thenicotinamide adenine dinucleotide, while the descriptor “NAD(P)H₂”indicates the reduced state of the phosphorylated and non-phosphorylatedforms of the nicotinamide adenine dinucleotide. Further, the descriptors“1,2-NAD(P)H₂”, “1,4-NAD(P)H₂” and “1,6-NAD(P)H₂” are used to indicatethe three stereoisomers of the nicotinamide ring that can occur uponreduction of NAD(P), in which a hydrogen molecule has been formallyadded across the 1,2-, 1,4- and 1,6-positions of the nicotinamide ring.It will be clear to those skilled in the art that 1,4-NAD(P)H₂ iscommonly called β-NADH, and is the stereoisomer of the reduced state ofthe phosphorylated and non-phosphorylated forms of nicotinamide adeninedinucleotide which is active with oxidoreductase and P450 enzymes.

In the present disclosure, the 4,4′-dimer that is also known to form asa consequence of a one-electron transfer to NAD(P) is indicated by thedescriptor “[NAD(P)]₂”.

The present disclosure, in some embodiments, is directed to an improvedversion and improved use of the “Electrochemical Bioreactor Module”(EBM) previously described in PCT Patent Application Publication Nos.WO2014039767 A1 and WO2016070168 A1, which are incorporated herein byreference in its entirety.

An EBM can include one or more of the following components:

-   -   a. an anode contained in an anode chamber and a cathode        contained in a cathode chamber;    -   b. deionized water in the anode chamber in contact with the        anode;    -   c. a proton permeable membrane that separates the anode and        cathode chambers;    -   d. a liquid phase in the cathode chamber continuously in contact        with the cathode, said liquid phase optionally comprising an        electron transfer mediator (ETM) capable of transferring        reducing equivalents to a redox enzyme system, said redox enzyme        system comprising a redox enzyme and a cofactor;    -   e. a process stream containing a substrate to be transformed via        catalysis by the redox enzyme system into a desired product;    -   f. a membrane located between the cathode and the process        stream, said membrane capable of preventing the optional ETM and        the redox enzyme system from significantly entering into the        process stream; and    -   g. an external power source providing a voltage between the        anode and the cathode.

In certain embodiments, an improved EBM or process using the EBM of thepresent disclosure can include the enzyme renalase, the Mung Bean PhenolOxidase, and illumination to recover the undesired forms of cofactorthat result from electrochemical reduction of NAD(P).

The enzyme renalase (Moran et al, J. Am. Chem. Soc. (2013), 135,13980-13987; Moran, G. R., Biochimica et Biophysica Acta 1864 (2016)177-186), is capable of oxidizing the 1,2-dihydro-NAD and1,6-dihydro-NAD species back to the oxidized state of the nicotinamideadenine dinucleotide cofactor.

Another enzyme isolated from Mung Bean and currently termed “Mung BeanPhenol Oxidase” (Fricks et al, Arch. Biochem. Biophys, (1973)169,837-841). This enzyme oxidizes the 4,4′-dimer, [NAD(P)]₂, that can beformed under electrochemically reducing conditions back to the oxidizedstate of nicotinamide adenine dinucleotide. This enzyme has also beenreported as having the same activity as renalase and being able tooxidize the 1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ species back to the oxidizedstate of nicotinamide adenine dinucleotide as well (Kono et al, Bull.Agr. Chem. Soc. Japan, (1958) 22(6) 404-410).

It has also recently been shown that illumination of electrochemicallyprepared 4,4′-dimer at about 254 nm or at wavelengths exceeding 320 nmleads to regeneration of the oxidized state of nicotinamide adeninedinucleotide. (Czochralska et al, (1980) Arch. Biochem. Biophys. 199,497; Czochralska et al, (1990) Photochem. Photobiol. 51, 401-410).

Thus it is useful to incorporate the renalase and Mung Bean PhenolOxidase enzymes, and the illumination of the [NAD(P)]₂ dimer ofnicotinamide adenine dinucleotide in a process for the recovery ofcofactor material, and for the prevention of the loss of cofactormaterial to forms which are not used by oxidoreductase enzymes such asdehydrogenases, ketoreductases or P450 enzymes.

In one embodiment, the present disclosure provides a process thatcomprises:

-   -   a) a process stream containing NAD(P) which is passed through        the cathode chamber of an electrochemical cell such that the        desired, biologically active 1,4-NAD(P)H₂ is produced, together        with the undesired 1,2-NAD(P)H₂, 1,6-NAD(P)H₂ and [NAD(P)]₂        species;    -   b) providing a substrate molecule that is to be transformed via        the catalytic reaction of an oxidoreductase enzyme, such as a        dehydrogenase or ketoreductase, or a P450 enzyme, to produce a        desired product molecule;    -   c) contacting the process stream containing both the desired and        undesired cofactor species with an oxidoreductase, such as a        dehydrogenase or ketoreductase, or a P450 enzyme, capable of        using the reducing equivalents presented by the 1,4-NAD(P)H₂ to        perform a desired reaction in which a substrate molecule is        transformed to a desired product molecule, while the        1,4-NAD(P)H₂ is concomitantly transformed back to NAD(P);    -   d) recovering the cofactor material from the undesired, leaving        the undesired 1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ cofactor species        present in the process stream by contacting the process stream        with the renalase enzyme in the presence of oxygen for a        sufficient time that the renalase enzyme can catalyze the        oxidation of the undesired 1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ species        that may be present in the process stream to the oxidized form        of the cofactor, NAD(P);    -   e) recovering the cofactor material from the undesired and        [NAD(P)]₂ species present in the process stream by contacting        the process stream with Mung Bean Phenol Oxidase in the presence        of oxygen for a sufficient time that the Mung Bean Phenol        Oxidase enzyme can catalyze the oxidation and any [NAD(P)]₂        present in the process stream to the oxidized form of the        cofactor NAD(P);    -   f) returning the process stream now containing all of the        original nicotinamide adenine dinucleotide as the oxidized form        NAD(P) to the cathode chamber of the electrochemical cell for        reduction.

In one embodiment, a process stream containing NAD(P) is passed throughthe cathode chamber of the electrochemical cell such that 1,2-NAD(P)H₂,1,4-NAD(P)H₂, 1,6-NAD(P)H₂ and the 4,4′-dimer, [NAD(P)]₂ are produced.The process stream containing the mixture of NAD(P)H₂ species is thencontacted with an oxidoreductase enzyme (e.g. a dehydrogenase,ketoreductase, or P450 enzyme) capable of using the reducing equivalentsfrom the 1,4-NAD(P)H₂ to perform a desired reaction in which a substratemolecule is transformed to a desired product molecule, while the1,4-NAD(P)H₂ is concomitantly transformed back to NAD(P). The enzymeutilizing the 1,4-NAD(P)H₂ may be present in the process stream,immobilized on beads or other suitable material and held in a packedcolumn, or contained by a membrane as described in PCT PatentApplication Publication No. WO2016070168 A1, which is incorporatedherein by reference in its entirety.

The process stream, after contacting the oxidoreductase enzyme and afterthe 1,4-NAD(P)H₂ has been transformed back to NAD(P), is then contactedwith the renalase enzyme. In the presence of oxygen, the renalase enzymeoxidizes the undesired 1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ species that may bepresent back to NAD(P) with the concomitant production of hydrogenperoxide. An enzyme such as catalase, or a suitable inorganic catalyst(e.g. MnO₂), may be present to decompose the hydrogen peroxide and thusprevent deleterious effects by the hydrogen peroxide.

The renalase enzyme may be immobilized on beads or a suitable support,immobilized on, or contained by, a membrane or kept from freelycirculating in the process stream by other standard methods known tothose skilled in the art of enzymatic reactions. As the renalase enzymehas some activity towards the desirable 1,4-NAD(P)H₂ species and willoxidize this back to NAD(P), it is better if the renalase enzyme is notfreely circulating in the process stream, and that the process stream iscontacted with the renalase after the 1,4-NAD(P)H₂ present in theprocess stream has been consumed by the oxidoreductase enzyme, and hasbeen used to perform a useful reaction on a substrate.

The process stream is then brought into contact with the Mung BeanPhenol oxidase in the presence of oxygen and any undesired 4,4′-dimer,[NAD(P)]₂, present is thus oxidized back to NAD(P) with the concomitantproduction of hydrogen peroxide. The Mung Bean Phenol Oxidase may beimmobilized on beads or a suitable support, immobilized on, or containedby, a membrane or kept from freely circulating in the process stream byother standard methods known to those skilled in the art of enzymaticreactions. As the Mung Bean Phenol Oxidase enzyme has some activitytowards the desirable 1,4-NAD(P)H₂ species and will oxidize this back toNAD(P), it is better if the Mung Bean Phenol Oxidase enzyme is notfreely circulating in the process stream, and that the process stream iscontacted with the Mung Bean Phenol Oxidase after the desirable1,4-NAD(P)H₂ present in the process stream has been consumed by theoxidoreductase enzyme.

In another embodiment, the process stream, after contacting theoxidoreductase enzyme, may be contacted first with the Mung Bean PhenolOxidase enzyme for oxidation of the undesired [NAD(P)]₂ back to NAD(P),and then subsequently with the renalase enzyme for oxidation of theundesired 1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ species back to NAD(P).

In another embodiment, the process stream, after contacting theoxidoreductase enzyme may be contacted with the renalase enzyme andsubsequently irradiated with light at 254 nm or at wavelengths exceeding320 nm for decomposing the undesired [NAD(P)]₂ dimer back to NAD(P).

In another embodiment, the process stream, after contacting theoxidoreductase enzyme, may be contacted not only with the Mung BeanPhenol Oxidase enzyme for oxidation of the undesired [NAD(P)]₂ dimerback to NAD(P), and also for oxidation of the undesired 1,2-NAD(P)H₂ and1,6-NAD(P)H₂ species to NAD(P) as the Mung Bean Phenol Oxidase enzyme isknown to have activity for the oxidation of the undesired 1,2-NAD(P)H₂and 1,6-NAD(P)H₂ species back to NAD(P).

It will be clear to those skilled in the art that the order of the stepsin the process for recovering cofactor material as NAD(P) from theundesired 1,2-NAD(P)H₂, 1,6-NAD(P)H₂, and [NAD(P)]₂ species by the abovedescribed steps of contacting the process stream with renalase, MungBean Phenol Oxidase or illuminating it with light at 254 nm or atwavelengths exceeding 320 nm may be done in any order or combination.

It will be clear to those skilled in the art that the Mung Bean PhenolOxidase and the renalase enzymes may be engineered by known methods toallow greater stability and therefore lifetime in the process, or toallow greater activity, or to allow increased ease and efficiency ofimmobilization, or to allow the more efficient expression and isolationof these enzymes.

It will be equally clear to those skilled in the art of enzymaticreactions that the Mung Bean Phenol Oxidase enzyme may be engineered toallow increased activity for the oxidation of the undesired 1,2-NAD(P)H₂and 1,6-NAD(P)H₂, species to NAD(P), in addition to its activity foroxidizing the undesired [NAD(P)]₂ dimer to NAD(P).

In certain embodiments, the renalase enzyme and the Mung Bean PhenolOxidase enzyme may be engineered to have insignificant (e.g., lower thanwide-type) activity on the desirable 1,4-NAD(P)H₂ species, but useful(e.g., higher than wide-type) activity on the undesired 1,2-NAD(P)H₂,1,6-NAD(P)H₂, and [NAD(P)]₂ species resulting from the electrochemicalreduction of NAD(P). These enzymes may then be circulated with theprocess stream, or the enzymes may be immobilized or otherwise containedand contacted with the process stream in any order that is convenient,including prior to contacting the process stream with the oxidoreductaseor P450 enzyme which is acting upon the provided substrate.

EXAMPLES Example I

This example describes an exemplary process in which the undesired1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ isomers are re-oxidized to NAD(P) byrenalase, and the undesired [NAD(P)]₂ dimer is oxidatively cleaved toNAD(P) by Mung Bean Phenol Oxidase.

To demonstrate the process described in the present disclosure, MungBean Phenol Oxidase is extracted from Mung Bean seedlings as describedby Burnett and Underwood (Biochem. 7(10), 3328-3333, 1968). The genehuman renalase isoform 1 is cloned onto a plasmid for expression in E.coli, including codon optimization. The sequence of the renalase isrevealed by Pandini et al. (Protein Expression and Purification 72(2010) 244-253). The enzyme is expressed as an inclusion body, theinclusion bodies collected and refolded as described by Padini et al(Protein Expression and Purification 72 (2010) 244-253). The renalaseenzyme (MW=35 KDa) is held in a container in which an inlet and anoutlet are provided, with a permeable membrane between the inlet and theoutlet that is of a sufficiently low molecular weight cutoff to preventthe renalase from passing through it, but also having a sufficientlyhigh molecular weight cutoff that all species of the nicotinamideadenine dinucleotide present in the process stream can pass through it.A membrane with a molecular weight cutoff of 10 KDa is used. In asimilar manner, the Mung Bean Phenol Oxidase enzyme is held in aseparate container with an inlet and an outlet and that also includes amembrane between the inlet and the outlet that is permeable, having asufficiently low molecular weight cutoff that the Mung Bean PhenolOxidase cannot pass through it, but a sufficiently high molecular weightcutoff that all species of the nicotinamide adenine dinucleotide presentin the process stream can pass through it. A membrane with a molecularweight cutoff of 10 KDa is used. To allow the use of an oxidoreductaseenzyme that demonstrates the presence of useful amounts of the desired1,4-NAD(P)H₂ cofactor, the membrane system described in published PCTPatent Application WO2016070168 A1 published 6 May 2016, and which isalso described in U.S. Pat. Nos. 4,705,704 and 5,077,217 for the purposeof containing an enzyme. The enzyme Horse Liver Alcohol Dehydrogenase isused, with cyclohexanone or benzaldehyde used as the substrate for thealcohol dehydrogenase enzyme. A process stream containing 1 to 2 mM ofNAD(P) is prepared and buffered to a pH suitable for all three enzymes;a pH of 7.5 is used. The process stream containing the NAD(P) is pumpedthrough the cathode chamber of the electrochemical cell, which isenergized at approximately 2 volts, and the NAD(P) cofactor is reducedto give the desired 1,4-NAD(P)H₂ form of the cofactor, and also theundesired 1,2-NAD(P)H₂, 1,6-NAD(P)H₂ and 4,4′-dimer, [NAD(P)]₂ forms ofthe cofactor. Exiting the cathode chamber, the NAD(P) process streamgoes through the membrane system containing the alcohol dehydrogenase,where the desired 1,4-NAD(P)H₂ form of the cofactor provides reducingequivalents to the alcohol dehydrogenase, the cyclohexanone orbenzaldehyde substrate is reduced to cyclohexanol or benzyl alcoholproduct respectively, thus consuming the 1,4-NAD(P)H₂ form of thecofactor and producing the oxidized form NAD(P). The process stream thenproceeds to the inlet of the container holding the renalase enzyme. Theprocess stream mixes with the renalase enzyme and the renalase enzymeoxidizes the undesired 1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ forms of thecofactor to NAD(P). The process stream then passes through the membranebut the renalase enzyme is held in its container by the membrane. Afterpassing through the membrane the process stream proceeds to the outletof the container holding the renalase enzyme. From here the processstream passes to the inlet of the container holding the Mung Bean PhenolOxidase enzyme. The process stream mixes with the Mung Bean PhenolOxidase enzyme and the enzyme oxidizes the undesired 4,4′dimer,[NAD(P)]₂ form of the cofactor to NAD(P). The process stream then passesthrough the membrane but the Mung Bean Phenol Oxidase enzyme is held inits container by the membrane. The process stream exits through theoutlet of the container holding the Mung Bean Phenol Oxidase enzyme, andnow contains only NAD(P) as the other forms of the cofactor have allbeen oxidized by the alcohol dehydrogenase, the renalase and the MungBean Phenol Oxidase enzymes. The process stream is returned to the pump,and sent into the cathode chamber to repeat the cycle.

Example II

This example describes an exemplary process in which the undesired1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ isomers are re-oxidized to NAD(P) byrenalase, and the undesired [NAD(P)]₂ dimer is oxidatively cleaved toNAD(P) by illumination with UV light at 254 nm.

The process described in Example I is used, excepting the containerholding the Mung Bean Phenol Oxidase is replaced by a quartz tubethrough which the process stream can flow unimpeded by any membrane.Outside the quartz tube an ultraviolet lamp is present, and illuminatesthe tube with light at 254 nm which passes through the quartz andphotolytically cleaves the 4,4-dimer of the cofactor, [NAD(P)]₂, toNAD(P). Upon exiting the quartz tube, the process stream is returned tothe pump, and sent into the cathode chamber to repeat the cycle.

EQUIVALENTS

The present disclosure provides among other things novel methods anddevices for providing reducing equivalents to biological systems. Whilespecific embodiments of the subject disclosure have been discussed, theabove specification is illustrative and not restrictive. Many variationsof the disclosure will become apparent to those skilled in the art uponreview of this specification. The full scope of the disclosure should bedetermined by reference to the claims, along with their full scope ofequivalents, and the specification, along with such variations.

INCORPORATION BY REFERENCE

All publications, patents and patent applications cited above areincorporated by reference herein in their entirety for all purposes tothe same extent as if each individual publication or patent applicationwere specifically indicated to be so incorporated by reference.

1. A system for electrochemically generating NAD(P)H₂ reducingequivalents comprising: an electrochemical cell comprising an anodecontained in an anode chamber and a cathode contained in a cathodechamber; a first process stream containing NAD(P), passing through thecathode chamber and continuously in contact with the cathode from whichelectrons are transferred to the NAD(P) to produce a second processstream containing reduced species 1,4-NAD(P)H₂, 1,2-NAD(P)H₂,1,6-NAD(P)H₂, and [NAD(P)]₂, while optionally producing hydrogen; asubstrate of an oxidoreductase or P450 enzyme, which, when contactedwith the second process stream in the presence of the oxidoreductase orP450 enzyme, is transformed to a product while concomitantly consumingthe 1,4-NAD(P)H₂ in the second process stream and producing a firstrecovered NAD(P) and a third process stream; and at least one of a MungBean Phenol Oxidase enzyme and illumination at a wavelength of about 254nm or exceeding about 320 nm, which, when contacted with the thirdprocess stream, convert at least one of the 1,2-NAD(P)H₂, 1,6-NAD(P)H₂,and [NAD(P)]₂ therein to a second and optionally a third recoveredNAD(P).
 2. The system of claim 1, comprising the Mung Bean PhenolOxidase enzyme for converting the 1,2-NAD(P)H₂, 1,6-NAD(P)H₂, and/or[NAD(P)]₂ into the second recovered NAD(P) and/or the third recoveredNAD(P).
 3. The system of claim 1, comprising the illumination forconverting the [NAD(P)]₂ into NAD(P).
 4. The system of claim 1,comprising the Mung Bean Phenol Oxidase enzyme and the illumination. 5.The system of claim 4, wherein the third process stream is contactedwith the Mung Bean Phenol Oxidase enzyme resulting in conversion of the1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ into the second recovered NAD(P) and afourth process stream, wherein the fourth process stream is contactedwith the illumination to convert the [NAD(P)]₂ therein to the thirdrecovered NAD(P).
 6. The system of claim 1, further comprising, in oneor more of the process streams, an electron transfer mediator (ETM)capable of transferring electrons to NAD(P).
 7. The system of claim 1,further comprising catalase for decomposing hydrogen peroxide producedby the Mung Bean Phenol Oxidase enzyme and/or the illumination.
 8. Thesystem of claim 2, wherein the Mung Bean Phenol Oxidase enzyme isrecombinantly expressed from microorganisms such as Escherichia coli,Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae,Pichia pastoris, and Bacillus megaterium.
 9. The system of claim 2,wherein the Mung Bean Phenol Oxidase enzyme is a mutant form having alower oxidation activity for 1,4-NAD(P)H₂ than the wild type Mung BeanPhenol Oxidase enzyme, and/or a higher oxidation activity for1,2-NAD(P)H₂, 1,6-NAD(P)H₂ and/or [NAD(P)]₂ than the wild type Mung BeanPhenol Oxidase enzyme, wherein preferably the mutant form isrecombinantly expressed from microorganisms such as Escherichia coli,Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae,Pichia pastoris, and Bacillus megaterium.
 10. A method forelectrochemically generating NAD(P)H₂ reducing equivalents comprising:a. providing an electrochemical cell comprising an anode contained in ananode chamber and a cathode contained in a cathode chamber; b. passingthrough the cathode chamber a first process stream which contains NAD(P)and is continuously in contact with the cathode from which electrons aretransferred to the NAD(P) to produce a second process stream containingreduced species 1,4-NAD(P)H₂, 1,2-NAD(P)H₂, 1,6-NAD(P)H₂, and [NAD(P)]₂,while optionally producing hydrogen; c. contacting the second processstream with a substrate of an oxidoreductase or P450 enzyme such thatthe substrate, in the presence of the oxidoreductase or P450 enzyme, istransformed to a product while concomitantly consuming the 1,4-NAD(P)H₂in the second process stream and producing a first recovered NAD(P) anda third process stream; and d. contacting the third process stream withat least one of a Mung Bean Phenol Oxidase enzyme and illumination at awavelength of about 254 nm or exceeding about 320 nm, thereby convertingat least one of the 1,2-NAD(P)H₂, 1,6-NAD(P)H₂, and [NAD(P)]₂ therein toa second recovered NAD(P) and optionally a third recovered NAD(P). 11.The method of claim 10, further comprising contacting the third processstream with the Mung Bean Phenol Oxidase enzyme for converting the1,2-NAD(P)H₂, 1,6-NAD(P)H₂, and/or [NAD(P)]₂ into the second recoveredNAD(P) and/or the third recovered NAD(P).
 12. The method of claim 10,further comprising contacting the third process stream with theillumination for converting the [NAD(P)]₂ into NAD(P).
 13. The method ofclaim 10, further comprising contacting the third process stream withthe Mung Bean Phenol Oxidase enzyme resulting in conversion of the1,2-NAD(P)H₂ and 1,6-NAD(P)H₂ therein into the second recovered NAD(P)and a fourth process stream, and further comprising contacting thefourth process stream with the illumination to convert the [NAD(P)]₂therein to the third recovered NAD(P).
 14. The method of claim 10,further comprising providing, in one or more of the process streams, anelectron transfer mediator (ETM) capable of transferring electrons toNAD(P).
 15. The method of claim 10, further comprising providingcatalase for decomposing hydrogen peroxide produced by the Mung BeanPhenol Oxidase enzyme, and/or the illumination.
 16. The method of claim10, wherein the Mung Bean Phenol Oxidase enzyme is recombinantlyexpressed from microorganisms such as Escherichia coli, Bacillussubtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichiapastoris, and Bacillus megaterium.
 17. The method of claim 10, whereinthe Mung Bean Phenol Oxidase enzyme is a mutant form having a loweroxidation activity for 1,4-NAD(P)H₂ than the wild type Mung Bean PhenolOxidase enzyme, and/or a higher oxidation activity for 1,2-NAD(P)H₂,1,6-NAD(P)H₂ and/or [NAD(P)]₂ than the wild type Mung Bean PhenolOxidase enzyme, wherein preferably the mutant form is recombinantlyexpressed from microorganisms such as Escherichia coli, Bacillussubtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichiapastoris, and Bacillus megaterium.
 18. The method of claim 10, furthercomprising returning the third process stream to the cathode chamber.